The proposed research involves investigations of the in vitro and in vivo metabolism of cyclophosphamide (CP) in an effort to define the role of metabolic activation in chemotherapy and toxicity of CP and understand the mechanisms by which CP produces specific depression of hepatic drug metabolism. Compounds to be studied include labeled and unlabeled CP and its metabolites (e.g., 4-hydroxy CP, 4-keto CP, carboxyphosphamide, phosphoramide mustard and other congeneres of CP), some inducers and inhibitors of CP metabolism, and specific drugs and anticancer agents used in conjunction with CP in the treatment and supportive care of cancer patients. The objectives of these studies are; (a) to elucidate the mechanism of action of CP and its interactions in vivo and in vitro with biological macromolecules; (b) to define the role of these interactions in chemotherapy and toxicity; and (c) to elucidate the mechanism by which CP causes specific depression of hepatic drug metabolism and evaluate the biological implications of this depression. Much of the proposed work will be performed (a) in vitro with 14(C-4)-CP, (3H-chloroethyl)-CP and some labeled metabolites of CP (e.g., 4-hydroxy CP, phosphoramide mustard), DNA (RNA or purine and pyrimidine-nucleoside polymers) and hepatic microsomes from mice and rats; (b) in vivo with labeled-CP in mice and rats, with and without tumors, to determine the in vivo macromolecules binding of CP and its relation to chemotherapy and toxicity; and (c) in vitro and in vivo with rats to elucidate th mechanism by which CP causes specific depression of hepatic drug metabolism; for the latter studies CP, various metabolites of CP and other alkylating agents will be tested. Particular methods to be used include: radiochemical techniques, thin layer chromatography, high pressure liquid chromatography, column chromatography, in vivo tumor chemotherapy, organic analyticall techniques and other chemical, biochemical and enzymological methods.